BASICS OF CONFOCAL IMAGING (PART I) INTERNAL COURSE 2012
LIGHT MICROSCOPY Lateral resolution Transmission Fluorescence d min 1.22 NA obj NA cond 0 0 rairy 0.61 NAobj Ernst Abbe Lord Rayleigh Depth of field (Axial resolution) d tot n NA 0 2 obj
IMAGING OF THICK SPECIMEN wide-field confocal out of focus light is bluring the image and thus reducing the contrast/resolution
OVERVIEW 1. History of confocal 2. Confocal principle 3. Main components 4. Important parameters
HISTORY OF CONFOCAL Historical overview First confocal First spinning disc First laser confocal
HISTORICAL OVERVIEW BC (before confocal) 1884 - spinning disk for image dissection (Nipkow) AD (after [confocal] development) 1957 - stage scanning confocal (Minsky) 1960 - invention of the laser 1968 - tandem-scanning confocal microscope (Egger, Petráň) 1980s - development of personal computer 1987 - commercial laser scanning confocal
FIRST CONFOCAL MICROSCOPE Transmission light Marvin Minsky, 1957
FIRST SPINNING DISC CONFOCAL Reflected light Egger, Petráň [Petrah nyu], 1968
CONFOCAL PRINCIPLE How it works Scanning mechanisms Modes of operation Confocal vs wide field
HOW IT WORKS Focal plane Out-of-focus plane Image plane Problem: Wide-field image contains significant amount of blur due to out-of-focus contribution
HOW IT WORKS Pinhole Out-of-focus plane Image plane Focal plane Problem: Wide-field image contains significant amount of blur due to out-of-focus contribution Solution: confocal or deconvolution
IMAGING POINT BY POINT Most of the light is rejected by the pinhole Intensive light source: laser Sensitive detector: PMT Scanner Computer
ILLUMINATION Wide-field Köhler illumination illumination source is in a fourier plane to the sample plane confocal -f f Critical illumination illumination source is in a conjugate plane to the sample plane -f f
MODES OF CONFOCAL Reflection Mostly used in materials sciences Useful for settings optimization Fluorescence Main mode for imaging in biology Also used in materials sciences
ADDITIONAL POSSIBILITIES Transmission For amplitude samples DIC For phase samples Laser polarized Koehler illumination important Additional detector required (PMT) No pinhole - images not confocal
TYPICAL LSCM SYSTEM
MAIN COMPONENTS Lasers Photomultiplier Spectral detection AOTF Filters, dichroics Objectives
LASERS Light Amplification by the Stimulated Emission of Radiation Gas laser Solid state laser Diode laser Diode pumped solid state laser Properties of laser-light Linear polarization Coherence (temporal and spatial) Narrow spectra (several nm) High intensity (up to several W)
TYPICAL LASER LINES Gas lasers: HeCd 442 Ar 364 488 514 ArKr 568 647 HeNe 543 594 612 Diode or DPSS 405 445 488 532 561 640 (More compact and easy to operate)
SUPERCONTINUUM FIBRE LASER In optics, a supercontinuum is formed when a collection of nonlinear processes act together upon a pump beam in order to cause severe spectral broadening of the original pump beam. The result is a smooth spectral continuum 20
PHOTOMULTIPLIER Quantum efficiency up to 12% Gain up to several millions Nonlinear gain on voltage
AVALANCHE PHOTODIODES Modest gain (500-1000) Substantial dark current high quantum efficiency (up tot 90 percent). Avalanche photodiodes are now being used in place of photomultiplier tubes for many low-light-level applications.
NEW CONFOCAL DETECTORS Hybrid detectors HyD (Leica) BIG detector (Zeiss)
SCANNING MECHANISM 24
XY SCANNING MECHANISM To obtain 2D image the specimen is scanned line by line Unidirectional or bidirectional scan Beam scanning is mostly used
Response SCANNING MECHANISM Galvanometer Hysteresis Current / A
SCAN MODE Uni-directional Bi-directional Amplitude=scan-field Frequency=scan-speed time
RESONANT SCANNERS linear galvanometers are limited in their scanning speed due to inertia typically range from 1 to 5 images per second faster resonant scanning galvanometer that vibrates at fixed frequency resonant frequency on the order of 4 to 8 kilohertz image capture rates on the order of 30 frames per second. 28
Z SCANNING MECHANISMS To obtain 3 D image of the specimen, it is necessary to move the excitation focus not only in XY direction but also in Z direction Move objective Z drive (large range, tens of nm accuracy). Zeiss, Olympus Piezo holder (small range but nm accuracy). PerkinElmer UV Move stage Piezo (high accuracy, but low stroke). Leica Galvo (large stroke, high accuracy). Leica
ACOUSTO OPTICAL TUNABLE FILTER (AOTF) Microsecond temporal resolution High transmission Intensity control possible Blanking outside scan area
FILTERS, DICHROICS PMT PMT AOTF instead of excitation filters Emission filters: LP, BP Dichroic (dichromatic): single, multiple Should match the fluorophore
SPECTRAL DETECTION Diffraction grating or prism decomposes fluorescence light into spectrum
CONFOCAL MICROSCOPE COMPONENTS Detection (Point Detectors) Photomultiplier GaAsP detectors (higher sensitivity) Scanner Galvanometric scanners Resonant scanners (high speed) Excitation Lasers AOTF (flexible, fast switching) Spectral components Filters, grating, prism, dichroic mirrors 33
LATERAL RESOLUTION NA=0.8 NA=1.4 1 1 0.1 0.1 0.01 0.01 1E-3 1E-3 1E-4 1E-4 1E-5 1E-5 1E-6 1E-6
Image LATERAL RESOLUTION Object
CONFOAL MICROSCOPY POINT SPREAD FUNCTION
Intensity CONFOAL MICROSCOPY POINT SPREAD FUNCTION Comparison Theoretical Point spread functions PSF (wide -field) PSF 2 (confocal) optical unit
HOW TO CHOOSE PINHOLE SIZE Pinhole is measured in Airy units 1 AU - diameter of the first minimum of the Airy disc Sectioning/signal optimum at 1 AU Pinhole diameter: d < 0.7 AU: optimal sectioning but very low signal 0.7 < d < 1 AU: some reduction in sectioning quality but significant increase in signal d > 1 AU: rapid decrease of optical sectioning quality
CHOICE OF OBJECTIVE Zeiss 63x/1.2 W Korr wd: 0.24 mm; thick specimen Zeiss 63x/1.4 Oil wd: 0.18 mm; thin specimen Match refractive index of sample and immersion media Use objectives with correction collar For confocal imaging objective magnification is not so important, only NA and degree of objective correction matters
SUMMARY Most of the light is rejected by the pinhole. Specimen with low florescence signal in widefield is very difficult to image in confocal. For confocal microscope objective NA and degree of correction is much more important than the magnification of the objective. Pinhole with the size of 1 AU is a good trade off between signal intensity and sectioning quality.